Partitioning in a two-polymer aqueous phase system was used to probe the surface properties of lymphoid cell subpopulations in aged male NZB/NZW F1 hybrid (B/W) mice, an important model of autoimmunity, immunodeficiency, and lymphoid malignancy. Spleen cells were fractionated by countercurrent distribution (CCD, a multiple-step extraction procedure) in a charged dextran-polyethylene glycol system. CCD of spleen cells from young, clinically normal male B/W mice yielded several broad distribution patterns which frequently had two or more peaks. Analysis of differentiation antigens and functional properties of cells from different parts of the distribution revealed a subfractionation of the three major lymphocyte subpopulations. B lymphocytes had a low partition coefficient (K); T cells had an intermediate K and null cells had the highest K. To examine the partitioning behavior of T lymphocytes, spleen cells which were nonadherent to nylon wool columns were subjected to CCD. Nonadherent cells from young B/W mice consistently gave a single peak with high K. Aged mice (18 months) usually had nonadherent cells with a predominantly low K. In some experiments a systematic increase in the number of these cells could be demonstrated with increasing mouse age. An analysis of the adherence and partitioning behavior of lymphocyte subpopulations revealed no change in the adherence properties or proportions of B lymphocytes in aged mice. The large proportion of cells having a low partition coefficient in the nonadherent spleen cell population of old mice appears to be due to an increase in the number of null cells and in a decrease in the K of some T lymphocytes.